L26/P-101 A biallelic splicing variant in WDR78 causes multiple morphological abnormalities of the sperm flagella

Biallelic Truncating Variant in LRGUK Is Associated With Severe Multiple Morphological Abnormalities of the Sperm Flagella and Sperm Nuclear Defects in Humans.

Multiple morphological abnormalities of the flagella (MMAF) is a severe form of male infertility characterized by immotile spermatozoa with absent, short, coiled, or irregular flagella. Despite advances in whole-exome sequencing, many cases remain genetically unexplained. Here, we identify a homozygous truncating variant in LRGUK (c.1063C>T; p.Arg355Ter) in an infertile man presenting with a typical MMAF phenotype. The variant, identified by whole-exome sequencing in a cohort of 168 MMAF patients and confirmed by Sanger sequencing, is predicted to result in loss of the C-terminal guanylate kinase-like domain. Immunofluorescence showed absence of LRGUK protein in patient spermatozoa, supporting a loss-of-function effect. Semen analysis revealed impaired motility and complete teratozoospermia. Morphological and ultrastructural analyzes demonstrated severe defects affecting both sperm head and flagellum, including disorganized axonemal architecture and central pair abnormalities. Nuclear analyzes showed increased nuclear size, defective chromatin compaction, and elevated DNA fragmentation. Immunostaining further indicated alterations of central apparatus-associated proteins, supporting a role for LRGUK in C1b projection organization. These findings identify LRGUK as a novel gene involved in male infertility, essential for spermatid morphogenesis and flagellum assembly.

A novel homozygous frameshift mutation in CFAP65 is associated with multiple morphological abnormalities of sperm flagella in a consanguineous Pakistani family

Multiple morphological abnormalities of the sperm flagella (MMAF) represent a severe form of asthenozoospermia that leads to male infertility, and it is commonly associated with genetic defects affecting flagellar components. Although the gene CFAP65 has been implicated in MMAF, its full mutational spectrum and clinical relevance within highly consanguineous populations remain poorly characterized. To elucidate the genetic basis of this condition, whole-exome sequencing followed by Sanger sequencing was performed in two infertile individuals from a consanguineous Pakistani family. Sperm morphology was assessed using hematoxylin-eosin (H&E) staining; while scanning and transmission electron microscopy (SEM and TEM) were utilized to evaluate ultrastructural defects. A novel homozygous frameshift mutation in CFAP65 (c.582_587delinsCG; p.Q194Hfs*4) was identified. This mutation introduces a premature stop codon within the transmembrane helix domain. Morphological analysis revealed classic MMAF features, including absent, short, bent, and coiled flagella in over 97% of sperm. TEM further demonstrated severe axonemal disorganization, with absent or disrupted microtubule doublets observed in 85% of the cross-sections. Based on clinical history and chest X-ray, the affected individuals reported no chronic respiratory symptoms and had no situs inversus, suggesting an isolated sperm-specific phenotype. In conclusion, this study identifies a novel domain-specific CFAP65 mutation associated with MMAF and male infertility in a consanguineous Pakistani family, thereby expanding the known genetic mutational landscape of MMAF. These findings reinforce the crucial role of CFAP65 in sperm flagellar morphogenesis and underscore its significance in the genetic diagnosis of male infertility.

A Novel Homozygous Mutation in ARL2BP Causes Multiple Morphological Abnormalities of the Flagella and Primary Ciliary Dyskinesia

Primary ciliary dyskinesia (PCD) and multiple morphological abnormalities of the sperm flagella (MMAF) frequently co‐occur in male infertility. However, the genetic basis of this syndromic presentation remains unclear. Using whole‐exome sequencing, we identified a novel homozygous ARL2BP splice‐site mutation (c.294‐2A>G) in a 23‐year‐old infertile male from a consanguineous family who presented with syndromic PCD and MMAF. This variant causes aberrant pre‐mRNA splicing and triggers nonsense‐mediated mRNA decay, resulting in the complete absence of ARL2BP protein expression. Transmission electron microscopy revealed extensive disorganization of flagellar axonemes with consistent central pair (CP) microtubule depletion and disorganization of peripheral doublets. Immunofluorescence confirmed a severe deficiency of the CP protein SPAG6 in the sperm flagella. Notably, the patient presented without retinal symptoms. Given that ARL2BP‐related retinitis pigmentosa generally emerges during the third decade, long‐term ophthalmological follow‐up is essential to detect delayed‐onset retinal degeneration. In conclusion, these findings confirm ARL2BP as a causative gene for both PCD and MMAF, expanding the genotypic and phenotypic spectrum of ciliopathies.

Identification of a Novel Splice-Site variant in TACR3 (c.888 + 1G > A) Associated with Asthenozoospermia and Hypogonadotropic Hypogonadism in an Iranian Family.

A Homozygous NUP210L Variant Is Associated With Severe Defects in Human Spermiogenesis.

BACKGROUND Spermiogenesis is a highly specialized differentiation process involving by extensive nuclear remodeling and cytoplasmic reorganization. Disruption of these events is a major cause of male infertility, however the genetic determinants underlying human spermiogenic failure remain incompletely understood. NUP210L, which encodes a testis-enriched nucleoporin, has recently been implicated in male infertility, although direct evidence in humans is limited and murine models do not fully recapitulate the human phenotype. OBJECTIVE To investigate the genetic basis and spermiogenic consequences of a homozygous NUP210L variant in a patient with severe oligoasthenoteratozoospermia (OAT), and to evaluate its impact on fertilization. MATERIALS AND METHODS A consanguineous family with an affected male proband was studied. Whole-exome sequencing followed by Sanger validation was performed to identify the causative variant. Comprehensive sperm analyses, including light and transmission electron microscopy, immunofluorescence, Western blotting, and chromomycin A3 staining, were conducted to assess ultrastructure, protein localization, and chromatin remodeling. Fertilization outcomes of intracytoplasmic sperm injection (ICSI) with or without artificial oocyte activation (AOA) were evaluated. RESULTS A homozygous in-frame deletion in NUP210L (c.1501_1503del, p.Ser501del) was identified. Patient sperm exhibited marked spermiogenic defects, including impaired chromatin condensation, acrosomal abnormalities, mitochondrial sheath disorganization, and fibrous sheath dysplasia. Although NUP210L protein levels were preserved, immunofluorescence revealed abnormal accumulation and mislocalization, suggesting functional impairment rather than loss of expression. Chromomycin A3 staining indicated significant histone retention, consistent with defective nuclear remodeling. Notably, the sperm-specific oocyte activation factor PLCζ was undetectable, consistent with total fertilization failure (TFF) following conventional ICSI. In contrast, ICSI combined with AOA successfully restored fertilization and enabled blastocyst development. CONCLUSION This study identifies a homozygous NUP210L variant associated with severe spermiogenic defects and impaired sperm function. The findings support a role for NUP210L in human spermiogenesis that is not fully recapitulated in murine models. The successful rescue of fertilization by AOA further suggests that NUP210L-related defects are associated with impaired oocyte activation, providing human-based evidence that nucleoporin dysfunction contributes to male infertility.

A synonymous NPR2 variant causes acromesomelic dysplasia through aberrant pre-mRNA splicing.

A homozygous synonymous NPR2 variant is identified in an individual with AMDM and aberrant splicing induced by a synonymous variant as a disease-causing mechanism affecting a core developmental signaling pathway is established.